Development of Primers and Probes for Genus and Species Specific Detection of ‘<i>Candidatus</i> Liberibacter Species’ by Real-Time PCR

Ananthakrishnan, G.; Choudhary, Nandlal; Roy, Avijit; Sengoda, Venkatesan G.; Postnikova, Elena; Hartung, John; Stone, Andrew L.; Damsteegt, V. D.; Schneider, William L.; Munyaneza, Joseph E.; Brlansky, R. H.

doi:10.1094/pdis-12-12-1174-re

Cited by 33 publications

(34 citation statements)

References 33 publications

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“…Liberibacter species in the plants and the psyllids, several DNA‐based detection methods have been developed. These include conventional and nested PCR (Hung et al , ; Liefting et al , ; Beard et al , ; Fujikawa et al , ) and real‐time PCR with or without probe (Li et al , , ; Teixeira et al , ; Ananthakrishnan et al , ; Beard et al , ; Bertolini et al , ; Nissinen et al , ; Teresani et al , ), and loop‐mediated amplification (LAMP) which produces a visible precipitate in the positive samples (Ravindran et al , ; Rigano et al , ). Wen et al () developed an SSR marker analysis PCR method for detection and differentiation of the haplotypes A and B of Ca .…”

Section: Disease Diagnosis Epidemics and Controlmentioning

confidence: 99%

Biology and epidemics of Candidatus Liberibacter species, psyllid‐transmitted plant‐pathogenic bacteria

Haapalainen

2014

Annals of Applied Biology

127

104

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Candidatus Liberibacter species are Gram-negative bacteria that live as phloem-limited obligate parasites in plants, and are associated with several plant diseases. These bacteria are transmitted by insects called psyllids, or jumping plant lice, which feed on plant phloem sap. Citrus huanglongbing (yellow shoot) or citrus greening disease is associated with three different species of Ca. Liberibacter -Ca. L. asiaticus, Ca. L. africanus and Ca. L. americanus -all originally found on different continents. Ca. L. asiaticus is the most severe pathogen, spread by Asian citrus psyllid Diaphorina citri and causing devastating epidemics in several countries. Ca. L. africanus occurs in Africa where it is spread by the African citrus psyllid Trioza erytreae. Ca. Liberibacter solanacearum is associated with diseases in several solanaceous plants, and transmitted by potato psyllid Bactericera cockerelli. Zebra chip disease is causing large damage in potato crops in North America. In Europe Ca. Liberibacter solanacearum is associated with diseases of the Apiaceae family of plants, carrot and celery, and transmitted by psyllids Trioza apicalis and Bactericera trigonica. When Ca. Liberibacter is suspected as the disease agent, the diagnosis is confirmed by DNA-based detection methods. Ca. Liberibacter-associated plant diseases can be controlled by using healthy plant propagation material, eradicating symptomatic plants, and by controlling the psyllid populations spreading the disease.

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Section: Disease Diagnosis Epidemics and Controlmentioning

confidence: 99%

Biology and epidemics of Candidatus Liberibacter species, psyllid‐transmitted plant‐pathogenic bacteria

Haapalainen

2014

Annals of Applied Biology

127

104

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“…The midribs of the leaves were excised and stored at -80°C until DNA extraction. DNA extraction was performed using DNeasy Plant Kits (Qiagen, Valencia, CA) as per the manufacturer's instructions and qRT-PCR was performed using rpoB primer (Ananthakrishnan et al, 2013):…”

Section: Methodsmentioning

confidence: 99%

Efficacy of In-field Thermotherapy in Comparison and Combination of Defoliation for Mitigating Huanglongbing in Sweet Orange

Vashisth¹,

Livingston²

2020

horts

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Huanglongbing (HLB), a bacterial disease, is one of the most destructive citrus diseases. For many decades, it has been known that using heat/high temperatures (thermotherapy) is effective in suppressing plant diseases, particularly the suppression of Candidatus Liberibacter (CLas, casual agent for HLB) when the treated plants are grown in pots (allowing treatment of roots) under controlled conditions. However, in-field thermotherapy has yielded inconsistent results: the tree shows vigorous and symptomless growth for a brief period and then relapses with HLB symptoms. To understand why, this 2-year study was conducted to evaluate the efficacy of in-field thermotherapy and its comparison with defoliation. A significant reduction in visible tree health, foliage, and yield was observed over time in all the treatments. The quantitative real-time polymerase chain reaction (qRT-PCR) results showed that in-field thermotherapy and other treatments were not effective in reducing CLas titer. Interestingly, the performance of thermotherapy and partial defoliated trees were comparable throughout the course of the experiment, indicating that the short-term effects and vigorous growth after thermotherapy are likely an artifact of defoliation and should not be confused with or considered recovery from disease. In an in-depth molecular and biochemical analysis, we found a few subtle transient (up to 15 days) differences occurring in the in-field thermotherapy trees. Genes associated with stress and plant defense response were observed to be altered by in-field thermotherapy. Overall, our results indicate that in-field thermotherapy is not an effective and reliable strategy for mitigating HLB in commercial citrus production and that its efficacy within in-field conditions is similar to manual defoliation. It is critical that any strategy aimed at mitigating HLB should target the whole plant, including the roots, as the CLas colonizes in shoots and the root system; therefore, it can possibly translocate within the entire plant with the bulk phloem flow.

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“…The TaqMan Universal PCR Master Mix (Life Technologies) was used for the DNA assay. DNA samples were analyzed with degenerate genus-specific (rpoB) primer/probe sets (Ananthakrishnan et al, 2013). The final concentration of qPCR mix contained 1X TaqMan Universal PCR Master Mix (10 mL), forward and reverse primers 2 mL each (600 nM each), probe 1 mL (300 nM), 5 mL of 4 ng/mL DNA sample (20 ng total), and nuclease free water for a total of 20 mL.…”

Section: Methodsmentioning

confidence: 99%

An Improved Method to Track Changes of Candidatus Liberibacter asiaticus Titer in HLB-affected Citrus Trees

Etxeberria¹,

González²,

Singerman³

et al. 2019

horts

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Monitoring the health of Huanglongbing-affected citrus trees by following changes in leaf Candidatus Liberibacter asiaticus (CLas) titer has an inherent element of imprecision because CLas titer varies considerably within the tree canopy and with calendar seasons. In addition, the destructive sampling method used to determine CLas titer entails a different set of leaves per sampling period adding to the inconsistency and inexactitude of the results. To overcome these ambiguities and to reduce the numerical variability between samples, we developed an experimental method that analyzes portions of the same treated leaves for up to four sampling periods. By assaying subsamples of adjacent locations of the same leaf, random variability was significantly reduced, and comparative analysis can be carried out with greater precision.

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Development of Primers and Probes for Genus and Species Specific Detection of ‘Candidatus Liberibacter Species’ by Real-Time PCR

Cited by 33 publications

References 33 publications

Biology and epidemics of Candidatus Liberibacter species, psyllid‐transmitted plant‐pathogenic bacteria

Biology and epidemics of Candidatus Liberibacter species, psyllid‐transmitted plant‐pathogenic bacteria

Efficacy of In-field Thermotherapy in Comparison and Combination of Defoliation for Mitigating Huanglongbing in Sweet Orange

An Improved Method to Track Changes of Candidatus Liberibacter asiaticus Titer in HLB-affected Citrus Trees

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